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Definitions

• the present invention relates to small RNAs (DDRNAs), inhibitors thereof, inhibitors of enzymes producing thereof, and their use to modulate the response of a cell to a DNA damaging event.
• DDRNAs small RNAs
• the invention concerns also a method to detect the presence or quantify DNA damage.
• the DNA damage response is a coordinate set of events that promptly follows the generation of a lesion in the DNA double helix. Detection of DNA discontinuities by specialized factors initiates a signaling cascade that, stemming from the site of DNA damage, amplifies the signal and reaches the whole nuclear space and the entire cell 1 . DDR signaling cascade initiation establishes a local self-feeding loop that leads to focal accumulation of upstream DDR factors in the form of cyto logically detectable DDR foci at damaged sites. Specifically, detection of a DNA double-strand break (DSB) triggers the activity of the protein kinase ATM that, among other factors, phosphorylates the histone variant H2AX ( ⁇ 2 ⁇ ) at the DNA damage site.
• DSB DNA double-strand break
• DDR activation can be triggered by exogenous DNA damaging agents such as ionizing radiations and chemotherapeutic agents (i.e. including but not limited to bleomycin) and by endogenous physiological events such as meiotic recombination, V(D)J recombination at the immunoglobulins and T cell receptor loci, telomere shortening and reactive oxygen species, as well as pathological events such as oncogene activation, viral integration in the genome, viral replication and bacterial infection ⁇ 82 .
• exogenous DNA damaging agents such as ionizing radiations and chemotherapeutic agents (i.e. including but not limited to bleomycin)
• endogenous physiological events such as meiotic recombination, V(D)J recombination at the immunoglobulins and T cell receptor loci, telomere shortening and reactive oxygen species, as well as pathological events such as oncogene activation, viral integration in the genome, viral replication and bacterial infection ⁇
• telomeres dysfunction and oncogene activation can generate a sustained DDR leading to a permanent cell-cycle arrest known as cellular senescence 2 .
• bacteria have been shown to generate persistent DNA damage and cellular senescence in mammals 82 .
• pathologies associated with altered telomere functions have been reported as "telomeropathies" 85 .
• ncRNAs non- coding RNAs
• RNA interference pathway RNA interference pathway
• RNA molecules including small interfering RNAs (siRNAs), microRNAs, repeat-associated small interfering RNAs (rasiRNAs), Piwi- interacting RNAs (piRNAs) 15 and QDE-2 interacting RNAs (qiRNA) in Neurospora crassa 16 .
• small interfering RNAs small interfering RNAs
• microRNAs microRNAs
• rasiRNAs repeat-associated small interfering RNAs
• piRNAs Piwi- interacting RNAs
• qiRNA QDE-2 interacting RNAs
• piRNAs and qiRNAs have been implicated in genome stability maintenance 16 and a family of microRNAs (miR-34) has been shown to act downstream of p53 19 . It is presently unknown whether any RNAs have any direct role in the control of DDR activation at sites of DNA damage.
• US2006105384 is focused on a technique for detecting and diagnosing disease conditions, as well as health conditions due to exposure to environmental conditions by detecting and identifying DNA or RNA damage markers. This technique is based on measurement of free levels of nucleotide excision products resulting from DNA or RNA damage.
• the DDRNAs of the instant invention are not nucleotide excision products.
• JP2009171895 concerns a method for analyzing the function of a non-coding RNA (ncRNA) existing in a nucleus by destroying the ncRNA by introducing an antisense oligo- molecule containing substantially the same sequence as a sequence complementary to a single-stranded region in the secondary structure of the target ncRNA to a cell nucleus and destroying the RNA molecule.
• ncRNA non-coding RNA
• WO2012/013821 relates to the field of cancer, particularly cancers wherein p53 tumour suppression function is lost or impaired. It is shown herein that Dicer is a synthetic lethal partner of p53, allowing the selective targeting and killing of cancer cells. The effects of Dicer on survival on cancer cells are mediated through the miR 17-92 cluster and inhibition of members of this miRNA cluster is an attractive treatment strategy in cancer. Most particularly, these findings are of importance in the field of retinoblastoma.
• WO2011/157294 relates to compositions comprising an inhibitor of a polynucleotide, said polynucleotide to be inhibited being capable of decreasing or suppressing expression of Dicer or a biologically active derivative thereof for use in treating or preventing cancer, metastasis, heart failure, cardiac remodelling, dilated cardiomyopathy, autoimmune diseases, or diseases or disorders related thereto. Furthermore, the present invention also relates to methods of treating or preventing cancer, metastasis, heart failure, cardiac remodelling, dilated cardiomyopathy, autoimmune diseases, or diseases or disorders related thereto. DDRNAs are not mentioned nor the impact of Dicer modulation on DNA damage related events and DDR modulation.
• WO2009/ 102225 relates to compositions and methods for cancer diagnosis, research and therapy, including but not limited to, cancer markers.
• the present invention relates to ncRNAs as diagnostic markers and clinical targets for prostate, lung, breast and pancreatic cancer.
• US2012289581 relates to long non-coding RNAs (IncRNAs) and methods of using them diagnostically and therapeutically for treatment of cancer, stem cell therapy, or regenerative medicine are disclosed.
• the invention relates to IncRNAs that play roles in regulation of genes involved in cell proliferation, differentiation, and apoptosis.
• Such IncRNAs can be used as bio markers to monitor cell proliferation and differentiation during cancer progression or tissue regeneration.
• PANDA a P21 -Associated NcRNA, DNA damage Activated
• Inhibitors of PANDA sensitize cancerous cells to chemotherapy and can be used in combination with chemo therapeutic agents for treatment of cancer.
• Limmer Ket al. (2013) used a Molecular Force Assay (MFA) to measure the activity of Dicer.
• MFA Molecular Force Assay
• RNA sequence that forms an aptamer-binding site for paromomycin, a 615-dalton aminoglycoside.
• Dicer activity is modulated as a function of concentration and incubation time: the addition of paromomycin leads to a decrease of Dicer activity according to the amount of ligand.
• the measured dissociation constant of paromomycin to its aptamer was found to agree well with literature values.
• the parallel format of the MFA allows a large-scale search and analysis for ligands for any RNA sequence.
• DDRNA of the instant invention have been characterized for distinct functions: DDRNAs control DDR signaling, whereas diRNA of Wei et al are not shown to have any role in DDR signaling: Wei et al show no evidence of altered DDR activation, as detected by nuclear DDR foci formation or of DDR proteins activation, for instance by phosphorylation, or of altered DNA damage checkpoint functions or modulation of cellular senescence. Thus there is no demonstrated overlap between their functions.
• DICER SUMMARY OF THE INVENTION DICER (Gene ID: 23405; Official Symbol: DICERl Name: dicer 1, ribonuclease type III [Homo sapiens] Other Aliases: DCR1, Dicer, HERNA, KIAA0928, MNG1; Other Designations: Dicerl, Dcr-1 homolog; K12H4.8-LIKE; dicer 1, double-stranded RNA25 specific endoribonuclease; endoribonuclease Dicer; helicase MOI; helicase with RNAse motif; helicase-moi, Chromosome: 14; Location: 14q32.13, Annotation: Chromosome 14, NC_000014.8 (95552565..95623759, complement), MIM: 606241, NCBI version May 4, 2012) and DROSHA (Gene ID: 29102; Official Symbol: DROSHA Name: drosha, ribonuclease type III [Homo sapiens],
• RNAi Components of RNAi are thought to have evolved to preserve genome stability from the attacks of viruses and mobile genetic elements.
• RNA products generated by DICER and DROSHA are involved in chromatin assembly in Schizosaccharomyces pombe, gene silencing and cancer.
• the DNA damage response (DDR) is a signaling pathway that arrests the proliferation of cells undergoing genotoxic events to preserve genome stability. So far, RNAi and DDR signaling pathways have not been demonstrated to directly interact.
• the authors show that oncogene-induced senescent cells, cells thus bearing oncogene-induced DNA damage and consequent DDR activation, require DICER and DROSHA to maintain DDR activation and cell-cycle arrest.
• DICER and DROSHA are also necessary to activate DDR upon exogenous DNA damage, and DDR checkpoint functions depend on the ribonuclease activity of DICER.
• DICER is required for irradiation-induced DDR activation in vivo in zebrafish.
• DDR foci stability is sensitive to RNase A treatment, and DICER- and DROSHA- dependent small RNA products are required to restore DDR foci in RNase A-treated cells.
• Study of DDR activation at a DNA double-strand break within a unique and traceable exogenous integrated locus reveals that DDR focus formation requires locus-specific RNA molecules.
• DDRNAs short or small RNAs
• DDRNAs act differently from microRNAs and canonical RNAi mechanisms because: ⁇ DDRNAs act without the need for any other cellular RNA (see the results obtained with gel-extracted RNA and synthetic RNAs in RNAse A-treated cells experiments).
• DDRNAs can have a sequence (LAC or TET repeats) that has no endogenous cellular transcripts match and still be biological active
• DDRNAs can act fast (in minutes) at room temperature in cells inhibited for transcription and translation (see the results obtained in RNAse A-treated cells experiments).
• DDRNAs small RNAs
• said small RNAs being generated by processing by DICER and/or DROSHA of a RNA transcript synthesized upon transcription of the damaged genomic locus, or impairing the production thereof .
• the method further comprising the step of: b) exposing said cell to a DNA damaging treatment.
• the DNA damaging treatment belongs to the group of: radiotherapy, chemotherapy (i.e. hydroxyurea treatment, bleomycine treatment), a treatment that impairs DNA repair or any genotoxic treatment. It is another object of the invention a method for sensitizing a cell damaged in at least one sequence specific genomic locus to the effect of a DNA damaging treatment, comprising the step of:
• DDRNAs small R As
• said small R As being generated by processing by DICER and/or DROSHA of a RNA transcript synthesized upon transcription of the damaged genomic locus, or impairing the production thereof
• step a) and step b) are performed in any order.
• the DNA damaging treatment is a radiotherapy.
• the radiotherapy is any ionizing radiation.
• the cell is damaged in at least one sequence specific genomic locus by a genotoxic event.
• the genotoxic event belongs to the group of: cell transformation, cellular senescence, oncogene activation, DNA replication stress, reactive oxygen species, ionizing radiation, chemotherapeutic agents (i.e. comprising but not limiting to bleomycin), telomere shortening, damaged telomere, recombination including V(D)J recombination at the immunoglobulins and T cell receptor locus, viral integration in the genome, viral infection and replication, bacterial infection.
• chemotherapeutic agents i.e. comprising but not limiting to bleomycin
• telomere shortening telomere shortening
• damaged telomere recombination including V(D)J recombination at the immunoglobulins and T cell receptor locus
• viral integration in the genome viral infection and replication, bacterial infection.
• DDRNAs is performed by a sequence specific inhibitor molecule.
• sequence specific inhibitor molecule is a sequence specific oligonucleotide.
• sequence specific inhibitor oligonucleotide is a LNA molecule .
• the step of impairing the production of said small RNAs is performed by inhibiting the cleavage and/or helicase activity of DICER and/or DROSHA.
• DROSHA is a specific siRNA.
• the cell is a mammalian cell.
• a human cell Preferably a human cell.
• the cell carries a sequence specific DNA damaged genomic locus.
• a pre-cancerous cell a cancer cell, a senescent cell, a cell with damaged telomeres or a viral infected cell.
• the senescent cell has critically short and/or damaged and/or dysfunctional telomeres.
• an inhibitor of small RNAs DDRNAs
• said small RNAs being generated by processing by DICER and/or DROSHA of a RNA transcript synthesized upon transcription of a sequence specific damaged genomic locus for medical use.
• the inhibitor is for use in the treatment of a condition induced by the sequence specific damaged genomic locus.
• the condition is cancer and/or aging and/or a viral infection.
• aging is associated with critically short and/or damaged and/or dysfunctional telomeres.
• the inhibitor is a sequence specific inhibitor molecule.
• said sequence specific inhibitor molecule is a sequence specific oligonucleotide.
• said sequence specific inhibitor oligonucleotide is a LNA molecule.
• the inhibitor is an inhibitor of DICER and/or DROSHA. Still preferably the inhibitor is a siRNA.
• composition comprising the inhibitor as defined above.
• the pharmaceutical composition comprises carriers, diluents and/or excipients.
• the composition may be administered by parenteral, oral, intravenous, intranasal, intramuscular route or any suitable route.
• the pharmaceutical composition mya be administered in any effective amount to elicit the desired therapeutic effect.
• the composition may be in any forms: solution, tablet, ointment etc.
• It is a further object of the invention a method to detect the presence of damage to DNA in a sequence specific genomic locus in a cell comprising the steps of:
• DDRNAs small RNAs
• It is a further object of the invention a method to identify the genomic location of a damage to DNA in a sequence-specific genomic locus in a cell comprising the steps of:
• DDRNAs small RNAs
• DDRNAs sequencing said isolated and/or purified small RNAs
• It is a further object of the invention a method to quantify the DNA damage in a specific genomic locus in a cell comprising the steps of: a) measuring the amount of small RNAs (DDRNAs), said small RNAs being generated by processing by DICER and/or DROSHA of a RNA transcript synthesized upon transcription of the damaged genomic locus in said cell;
• DDRNAs small RNAs
• It is a further object of the invention a method for the diagnosis and/or prognosis of a condition associated with and/or induced by the generation of DNA damage in at least one sequence specific genomic locus comprising:
• RNAs a) measuring the amount of small RNAs, said small RNAs being generated by processing by DICER and/or DROSHA of a RNA transcript synthesized upon transcription of the damaged genomic locus in said cell;
• condition associated with and/or induced by the generation of DNA damage in at least one sequence specific genomic locus is selected from the group consisting of: cancer, aging, viral infection. Still preferably aging is associated with critically short and/or damaged and/or dysfunctional telomeres.
• It is a further object of the invention a method for monitoring the efficacy of therapy directed to a condition associated with and/or induced by the generation of DNA damage in at least one sequence specific genomic locus in a subject comprising:
• DDRNAs small RNAs
• condition associated with and/or induced by the generation of DNA damage in at least one sequence specific genomic locus is selected from the group consisting of: cancer, aging, viral infection. Still preferably aging is associated with damaged telomeres.
• the proper control may be an untreated cell, a healthy cell or a cell at various time point during the therapy.
• It is a further object of the invention a method of screening for an agent able to inhibit small RNAs (DDRNAs), said small RNAs being generated by processing by DICER and/or DROSHA of a RNA transcript synthesized upon transcription of a damaged genomic locus in a cell comprising the step of measuring the amount of said small RNAs upon exposure of the cell to said agent, and comparing to a proper control.
• the proper control may be a cell treated with a reference compound or a non-treated cell.
• DDRNAs are small RNAs, with the potential to form double- stranded pairs, that are generated by processing by DICER and/or DROSHA of a sequence specific RNA transcript synthesized upon transcription of a damaged DNA locus.
• DDRNAs are small RNAs of a length between 10 and 50 nucleotides. For example of a length between 17 and 32 nucleotides. For example of a length between 20 and 25 nucleotides. For example of a length between 22 and 23 nucleotides.
• Said DDRNAs function by favoring the sequence-specific accumulation of DDR factors at specific sites of DNA damage and promote DDR signaling (i.e. comprising but not limiting to protein phosphorylation events).
• a critically short telomere is a telomere able to engage the DDR machinery due to its critical short length.
• a damaged telomere is a telomere carrying a DNA lesion able to engage the DDR machinery.
• a dysfunctional telomere is a telomere that due to its altered protein and/or nucleic acid structure engages the DDR machinery
• an oncogenic stress may be a genotoxic stress (i.e. comprising but not limiting to DNA lesions, impaired DNA replication forks progression) due to oncogene activation, amplification, gain of function mutation, increased levels and activity.
• a cell carrying a DNA damage is a cell whose DNA damage is not exogenously induced (i.e. a cell comprising but not limiting to critically short telomere and damaged telomere, oncogenic stress, oxidative DNA damage).
• Aging is associated with telomeric DNA damage and DDR activation 2 ' 84 .
• Genotoxic treatments commonly used in cancer therapy are treatments associated with the generation of DNA damage (i.e. comprising but not limiting to radiotherapy and chemotherapy).
• a radiotherapy is a therapy based on the exposure to ionizing radiation.
• An effective dose of ionizing radiation is a dose of ionizing radiation able to generate the desired outcome.
• the skilled person in the art using common routine techniques knows how to determine such dose.
• a senescent cell is a cell retaining persistent DDR activation (usually following oncogenic stress and/or telomere shortening/DNA damage).
• DDRNAs short RNAs
• control cell may be a non-damaged cell or a healthy cell.
• the analysis may be carried out by quantitative Reverse Transcriptase-PCR, northern blot hybridization, next generation sequencing (Illumina etc), ion torrent technology or by any other means available, appropriated and known to the skilled person in the art. It may be performed on a cell or the blood or other biological fluids. It can also be performed in tissue lysates.
• DDRNAs short RNAs
• control may be a non-damaged cell or a healthy cell.
• the analysis may be carried out by qRT-PCR, northern blot hybridization, next generation sequencing (Illumina etc), ion torrent technology or by any other means available, appropriated and known to the skilled person in the art. It may be performed on a cell or the blood or other biological fluids. It can also be performed in tissue lysates.
• RNA interference means and/or 2. Inhibiting the synthesis of DICER and/or DROSHA by RNA interference means and/or;
• an inhibitor of DICER and/or DROSHA is able to have at least one of the above activities.
• inhibitorting small RNAs means:
• DDRNAs biogenesis and/or processing by inhibiting DICER and/or DROSHA as described above and/or 2. preventing DDRNAs synthesis by preventing the transcription of the longer RNA precursor eventually cleaved by DICER and/or DROSHA and/or
• oligonucleotides comprise but are not limiting to locked nucleic acids (LNA), phosphorothioate modified oligonucleotides, 2'-0-methoxyethyl modified oligonucleotides, 2' O-Methyl modified oligonucleotides, methylphosphonates, morpholino oligonucleotides, LNA-DNA-LNA gapmer oligonucleotides, Chimeric 2'-0-methyl RNA-DNA gapmer, N3'-P5' Phosphoroamidate, 2'-fluoro-arabino nucleic acid, Phosphoroamidate Morpholino, Cyclohexene nucleic acid, Tricyclo-DNA, Peptide nucleic acid, Unlocked nucleic acid , Hexitol nucleic
• DDRNAs functions by modifying them by means i.e. of methylation, and/or
• DDRNAs such as phosphorylation, methylation etc etc.
• a DICER and/or DROSHA inhibitor is an agent or molecule able to display at least one DICER and/or DROSHA inhibiting function as described above (inhibiting the enzymatic activity of DICER and/or DROSHA, inhibiting the synthesis of DICER and/or DROSHA, destabilizing the proteins DICER and/or DROSHA, inhibiting DICER and/or DROSHA activity by the expression of DICER and/or DROSHA alleles with dominant negative functions and/or; targeting the genomic loci responsible for DICER and/or DROSHA synthesis).
• An inhibitor of DDRNAs is an agent or molecule able to display at least one DICER and/or DROSHA inhibiting function as described above (preventing their synthesis, preventing their proper localization in the cell to prevent their processing and/or functions, preventing their accumulation, preventing their functions, preventing them to act as they would and/or preventing any modification of DDRNAs).
• DICER or DROSHA knockdown by siRNA pools in WI38 human fibroblasts impairs pATM, pS/TQ, MDCl, but not ⁇ 2 ⁇ , foci assembly.
• Cells were irradiated (lOGy) and fixed 7h later, d.
• Histograms show percentage of WI38 cells positive for pATM, pS/TQ, MDCl and ⁇ 2 ⁇ foci. e. pATM, pS/TQ and MDCl, but not ⁇ 2 ⁇ , foci formation is impaired in D/CEif ⁇ -hypomorphic cells.
• Cells were irradiated (2Gy) and fixed 2h later, f. Histograms show the percentage of cells positive for DDR foci. Error bars indicate s.e.m. (n > 3). Differences (*) are statistically significant (p-value ⁇ 0.001).
• siGFP was used as control.
• Cells were irradiated (lOGy) and labeled with BrdU for 7 hours before fixation. Histograms show the percentage of BrdU- positive cells in not-irradiated (-) and in irradiated (+) cells, b. D/CEi? exo " 5 -hypomorphic cells have impaired Gl/S checkpoint. Cells were irradiated (2Gy) and labeled with BrdU for 2 hours before fixation.
• Histograms show the percentage of BrdU-positive cells in not- irradiated (-) and in irradiated (+) cells.
• DICER knocked-down cells have impaired G2/M checkpoint.
• shGFP is used as control, d.
• the table shows the percentage of cells in Gl , S and G2 phase of the cell cycle at different time points post IR.
• FIG. 3 Irradiation induces pATM and ⁇ 2 ⁇ nuclear accumulation in control but not in Dicer 1 morpholino-injected zebrafish embryos, a. Images illustrating the location of the sections from the head of 3 days post fertilization (dpi) WT (not injected) and Dicer 1-morpho lino injected zebrafish larvae stained for pATM and ⁇ 2 ⁇ before and after irradiation (12 Gy). Sections were stained with DAPI (blue) and pATM or ⁇ 2 ⁇ antibody (green), b. Immunoblot analysis of pATM and ⁇ 2 ⁇ accumulation in extracts from not irradiated and irradiated wild-type embryos or Dicerl morpholino-injected embryos.
• FIG. 4 Irradiation-induced DDR foci are sensitive to RNase A treatment and are restored by short and DICER RNA products
• a Irradiated HeLa cells (2 Gy) were treated with PBS (-) or RNase A (+) and probed for 53BP1, pATM, pS/TQ, MDCl and ⁇ 2 ⁇ foci. 53BP1 , pATM, pS/TQ and MDCl, but not ⁇ 2 ⁇ , foci are strongly reduced upon RNase A treatment, b. Histograms report the percentage of cells positive for DDR foci.
• Addition of gel-purified RNA in the size range of 20-35nt allows DDR foci reformation in RNase A-treated cells.
• Irradiation- induced 53BP1, pS/TQ and pATM foci are restored in RNase A-treated cells when incubated with RNA of wild-type (WT RNA) RKO cells but not with RNA of DICER exonS - hypomorphic (DICER exon5 RNA) RKO cells.
• tRNA was used as control. ⁇ 2 ⁇ foci were not affected, e. Histograms report the percentage of cells positive for DDR foci. Error bars indicate s.e.m. (n>4). Differences are statistically significant (*p-value ⁇ 0.001).
• FIG. 5 Site-specific DDR focus formation is RNase A-sensitive and can be restored by locus-specific RNA in a MRE11-RAD50-NBS1 complex-dependent manner
• NIH2/4 mouse cells experiencing I-Sce I-induced DSB next to a Lac-operator array (LacO array) display a 53BP1 (green) and ⁇ 2 ⁇ (magenta) focus colocalizing with the Cherry- Lac focus (red).
• 53BP1 green
• ⁇ 2 ⁇ magenta focus colocalizing with the Cherry- Lac focus
• 53BP1 green
• ⁇ 2 ⁇ magenta focus colocalizing with the Cherry- Lac focus
• 53BP1 but not ⁇ 2 ⁇ focus
• Histograms show the percentage of cells in which 53BP1 and Cherry-Lac foci co-localize.
• RNA purified from NIH2/4 rescues 53BP1 foci formation in a dose-dependent manner, c.
• Incubation of RNase A-treated cells with RNA purified from NIH2/4 expressing I-Sce I restores 53BP1 focus at the I-Sce I induced cut site, while RNA from NIH3T3 parental cells expressing I- Sce I does not.
• d e. RNA from NIH2/4 cells, or parental one, was used in RNase A-treated NIH2/4 cells to test 53BP1 or pATM focus reformation in the presence of the MRN inhibitor mirin.
• RNA from parental cells 800ng
• concentrations lng/ ⁇ - lfg/ ⁇ , ten-fold dilution steps
• Locus-specific synthetic RNAs down to a concentration of lOOfg/ ⁇ , but not control ones, allow site-specific DDR activation, b.
• RNAs generated by recombinant DICER processing of RNA generated in vitro by transcription of a DNA fragment carrying the central portion of the integrated locus, or a control one of similar length were tested to restore DDR focus formation at the DNA damage site in RNase A-treated NIH2/4 cells.
• RNAs were tested at the concentration of lng/ ⁇ mixed with 800ng of RNA from parental cells. Locus-specific DICER RNA products, but not control ones, allow site-specific DDR activation, c. The fraction of 22-23 nt vs total short RNAs at the locus decreases in DICER and DROSHA knockdown samples both in uncut and cut conditions.
• DICER or DROSHA inactivation in OIS cells allows escape from senescence and cell-cycle progression
• a. DICER, DROSHA knockdown by siRNA pools in OIS cells were evaluated by QRT-PCR. ATM knockdown by siRNA pool was evaluated by immunofluorescence
• siGFP was used as control. Error bars indicate s.e.m. (n > 3). Differences are statistically significant (p-value ⁇ 0.001).
• DICER-, DROSHA- and DDR-inactivated OIS cells by transfection with siRNA pools, re-express MCM2, a marker of chromosomal DNA replication, and pH3, a marker of entry into mitosis. Error bars indicate s.e.m. (n > 3). Differences are statistically significant (p-value ⁇ 0.001). d and g. DICER and DROSHA knockdown was evaluated by QRT-PCR e. 53BP1 knockdown is evaluated by immunofluorescence.
• DICER or DROSHA inactivation in OIS cells does not alter SAHF maintenance and does not decrease DDR protein levels but impairs their activation over a range of siRNA concentrations
• DICER or DROSHA were inactivated by transfection with siRNA pool in OIS cells. Cells were stained for H3K9me3 SAHF marker and for 53BP1. siGFP transfected cells are used as control.
• DICER or DROSHA inactivation affects 53BP1 foci without altering SAHF stability
• siGFP transfected cells are used as control. Vinculin is used as loading control, d-f.
• concentrations (10 fold difference) of siRNA pools against DICER or DROSHA in OIS cells impair DDR foci detection. Error bars indicate s.e.m. (n > 3). Differences are statistically significant (p-value ⁇ 0.05). Knockdown was evaluated by QRT-PCR.
• DICER, DROSHA or GW182/TNRC6A were inactivated by siRNA pool in OIS cells.
• DICER-, DROSHA- or GW182/TNRC6A-inactivated cells were stained for 53BP1, pATM and pS/TQ markers of activated DDR.
• GW182/TNRC6A inactivation has no detectable effect on DDR.
• Differences for DICER and DROSHA, but not GW182, are statistically significant (p-value ⁇ 0.005). Error bars indicate s.e.m. (n > 3).
• Knockdown was evaluated by QRT-PCR.
• FIG. 11 Simultaneous inactivation of TNRC6A/GW182, TNRC6B and TNC6C in OIS cells does not affect DDR foci formation while DROSHA inactivation does.
• TNRC6A, B and C simultaneous inactivation with either siRNA pools #1 or #2 has no detectable effect on DDR. Differences for DROSHA, but not TNRC6A, B and C, are statistically significant (p-value ⁇ 0.05). Error bars indicate s.e.m. (n > 3).
• siGFP transfected cells are used as control. Vinculin is used as loading control, d-g.
• DICER, DROSHA or GW182/TNRC6A was inactivated in Wi38 cells by siRNA pools, cells were irradiated (2Gy) and stained for 53BP1 10' later, for ⁇ 2 ⁇ , pATM and pS/TQ markers of activated DDR, 1 hour post IR.
• GW182/TNRC6A inactivation has no detectable effect on DDR. Differences for DICER and DROSHA, but not GW182, are statistically significant (p-value ⁇ 0.001). Error bars indicate s.e.m. (n > 3).
• c. Knockdown was evaluated by QRT-PCR.
• FIG. 17 Impaired DDR foci formation in D/CER ⁇ h pomorphic cells is rescued by wild type but not mutant DICER, a. Expression of wt DICER but not a mutant allele lacking endonuclease activity, restores DDR foci formation defect in DICER exon5 - hypomorphic RKO cells. 53BP1 foci formation was studied 10' after IR (2 Gy), pATM and pS/TQ 1 hour afterward, b. Histograms show the percentage of cells positive for the indicated DDR foci. Differences (*) are statistically significant (p-value ⁇ 0.001). Error bars indicate s.e.m. (n > 3).
• FIG. 18 ATM activation by autophosphorylation is impaired in DICER and DROSHA-inactivated cells
• a ATM activation following IR (10 Gy) is impaired in DICER and DROSHA knocked-down WI38 human fibroblasts as detected by immunoblot analysis for pATM. siGFP transfected cells are used as a positive control for ATM activation. Total ATM is unaffected as shown by vinculin.
• DICER knockdown was evaluated by immunoblot analysis
• DROSHA knockdown in WI38 cells was evaluated by QRT-PCR.
• ATM activation is impaired in irradiated (2 Gy) D/CEi ⁇ -hypomorphic R O cells. Total ATM and vinculin are used as loading control.
• DICER, DROSHA and 53BP1 knockdowns by siRNA pools in WI38 cells were monitored by QRT-PCR (a), immunoblot (b) and immunofluorescence (c), respectively, d, e.
• DICER-inactivated MRC-5 cells have impaired Gl/S checkpoint post IR (10 Gy).
• siGFP is used as control
• Figure 20 Loss of Gl/S and G2/M checkpoint activation in DICER knocked-down cells
• b Loss of Gl/S and G2/M checkpoint activation in DICER knocked-down cells
• DICER mutants (DICER-fiag, DICERl lOab-fiag and DICER44ab-fiag) in RKO cells
• DICER knockdown by shRNA in HEK293 cells was monitored by QRT- PCR.
• Dicerl morpholino-injected zebrafish embryos downregulate the expression of miRNAs.
• RFP Red Fluorescent Protein
• Dicerl morpholino 3 larvae on the right.
• the same embryos visualized by epifluorescence show an increase in RFP expression in Dicerl morpholino injected embryos, b. Specificity of miR126 sensor.
• Lamin A/C is used as loading control, d.
• RNase A affects 53BP1 and MDC1 but not DH2AX in the same focus.
• Irradiated HeLa cells were treated with PBS or RNase A.
• 53BP1 foci (green) and MDC1 foci (red) are affected by RNase A treatment while ⁇ 2 ⁇ (magenta) foci in the same cell are not.
• FIG 241 Ppo I-induced DDR foci are RNase A sensitive and reform upon RNA addition.
• Figure 251 Short RNAs promote DDR foci reformation at the DNA damage site a. Relative enrichment of miR-21 RNA compared to ⁇ -actin mRNA quantity evaluated by QRT-PCR in total RNA and short RNA-enriched fractions, b. Histograms show the percentage of cells positive for 53BP1, pATM and pS/TQ foci in irradiated HeLa cells, RNase A-treated, and cells incubated with 200ng of total RNA or a proportional volume of short RNA-enriched ( ⁇ 200nt) fraction. tRNA (200ng) was used as control, c. Irradiation- induced 53BP1 (green) and pATM (red) foci are restored in RNase A-treated cells by incubation with total and short RNAs-enriched fraction. tRNA was used as control.
• FIG. 261 Irradiation-induced DDR foci are restored in RNase A-treated cells by incubation with RNAs extracted from gel in the 20-35nt range
• b. QRT PCR analysis of RNU19 (200 nt), RNU44 (61 nt) and mir-21 (22 nt) in the indicated RNA fractions extracted from gel. Error bars indicate s.d. (n 3). Differences are statistically significant by student's t-test (p-value ⁇ 0.005).
• RNA extracted from D/C R ⁇ hypomorphic cells transfected with wild type but not mutant DICER allows DDR foci reformation in RNase A-treated cells
• R A extracted from shDICEPv-transfected (or GFP as control) HEK293 cells does not restore irradiation- induced 53BP1 foci in RNase A-treated HeLa cells. tRNA is used as negative control, c.
• Histograms report the percentage of cells positive for 53BP1 foci. d. Immunoblotting shows DICER knockdown efficiency, e. Histograms show the percentage of cells positive for 53BP1, pATM and pS/TQ foci in irradiated HeLa cells after RNase A treatment and incubation with RNA purified from siGFP and siDROSHA transfected cells. tRNA was used as control.
• FIG. 291 MRN complex involvement in DDR foci reformation after RNase treatment, a. MRN complex recruitment to the I-Sce I-induced DSB is sensitive to RNase A treatment. 53BP1, pATM and MREl 1 foci, but not ⁇ 2 ⁇ , are lost in RNase A treated NIH2/4 cells. Histograms show the percentage of cells in which 53BP1, pATM, MREl 1 or ⁇ 2 ⁇ foci colocalizing with the Cherry-Lac focus, b. Mirin impairs pATM activation on the I-Sce I-induced DSB. Histogram shows the percentage of cells in which pATM focus colocalize with the Cherry-Lac focus. Error bars indicate s.e.m. (n>3). Differences are statistically significant (*/?-va/we ⁇ 0.05).
• Identification of biologically active locus-specific molecules. Chemically synthesized locus-specific RNAs and in vitro generated DICER RNA products promote DDR focus formation at the DNA damage site in RNase A-treated cells, a.
• RNA shorter than 200 nt was purified and analyzed on the small RNA Bioanalyser kit (Agilent), b. Short RNA library was prepared and extracted from 6% polyacrylamide gel (indicated by an arrow at 100 bp), c. The integrity of the prepared library was checked using the Agilent DNA high sensitivity kit. d. Length distribution of tags in the library, e. Length distribution of tags in the library mapping to the exogenous integrated locus combining tags from cut and uncut samples, f. A pool of chemically synthesized oligonucleotides mapping to the exogenous locus was tested to restore DDR focus formation in RNase A-treated NIH2/4 cells.
• RNAs were tested at the concentration of lng/ ⁇ mixed with 800ng of tRNA. Locus-specific DICER RNA products, but not control ones, allow site-specific DDR activation at the DNA damage site. Histograms report the percentage of cells positive for DDR foci.
• Figure 311 Library profile and length distribution of selected sequenced samples, a. Bioanalyser profile of ⁇ 200 nt RNA from wildtype cut sample, b. Short RNA libraries were prepared from 40 ng RNA from each sample and run on a 6% PAGE gel. Arrow shows the 100 bp library band of interest, c. Wildtype cut library profile. Gel extracted libraries were run on Bioanalyser high sensitivity kit. Sequencing was performed on Hi seq Version 3. d-i. Tag length distribution of each library.
• FIG. 321 Dicer and Drosha knockdown downregulates miRNAs.
• Figure 331 Features of short RNAs arising from the locus, a. Length of tags arising from the locus before and after cut. Y-axis shows number of tags from the locus and X- axis depicts tag lengths in nucleotides. The bulk of short RNAs in wildtype samples before and after cut are in the 22-23 nt size range. Among knockdown samples, Dicer knockdown shows a broader tag length distribution, b. 22-23 nt percentage of the locus is significantly different from the same ratio of non miRNA genomic loci. Fractions of 22-23 nt vs total short RNAs at non miRNA genomic loci with at least 50 reads are shown in histograms with the vertical axis depicting their frequency.
• the vertical line depicts the ratio of 22-23 nt RNAs to the total at the locus.
• FIG. 34 Sequence-specific inhibitory oligonucleotides (i.e. LNAs) transfection impairs DDR at the locus in cut cells.
• the scheme (a) shows the TET-I-Scel-LAC locus, the DDRNAs generated upon cut (grey line), and the LNAs used (black dotted line).
• Cells were co-transfected with Cherry-Lac and I-Sce I-restriction endonuclease expressing vectors together with different sets (as in the legend in the figure) of LNA (200pM) with the potential to anneal to DDRNAs arising from the locus upon I-Scel-induced cleavage or a control LNA matching telomere sequence.
• FIG. 36 Sequence-specific inhibitory oligonucleotides (i.e. LNAs) transfection suppresses DDR and prevents BrdU reduction following telomere-uncapping.
• T19 fibrosarcoma cell line van Steensel, Cell 1998) was cultured in absence of doxycycline to induce expression of a dominant negative allele of TRF2 fused to flag. Induced cells are visualized by Flag immunostaining.
• Induced (Flag +) and uninduced (Flag -) cells were transfected with LNA molecules (200 nM) matching the sense (LNA 6), the antisense (LNA 5) telomeric sequence, and an unrelated (LNA 3, Cntrl) sequence at day 13 from induction, (a) 53BP1 foci-positive cells were scored at the indicated time points post transfection. LNA 5 and 6 cause a decrease in DDR-positive cells, to different extent, compared to control LNA in induced cells, while no difference was observed in uninduced cells. For the quantifications shown, around 30-100 cells were scored for each time point (b, c).
• Induced cells were incubated with BrdU for 16 hours and scored for BrdU incorporation 3 days following LNA trans fection.
• LNA 5 and 6 transfected Flag + cells show a significant increase in the percentage of BrdU-positive cells, compared to the Cntrl LNA (b), while no difference is observed in Flag - cells (c).
• BJ cells Early passage BJ cells, WI38 and MRC-5 (The American Type Culture Collection, ATCC) were grown under standard tissue culture conditions (37°C, 5% C0 2 ) in MEM supplemented with 10% fetal bovine serum, 1% L-glutamine, 1% non-essential aminoacids, 1% Na Pyruvate.
• HeLa, Phoenix ecotrophic and HEK293T cell lines were grown under standard tissue culture conditions (37°C, 5% C0 2 ) in DMEM, supplemented with 10% fetal bovine serum, 1% glutamine, 1% penicillin/streptomycin.
• R O, HCT116 and DLD1 colon cancer cell lines 25 were cultured in Mc'Coy 5 A medium +10% fetal calf serum, 1% penicillin/streptomycin.
• NIH2/4 35 where grown in DMEM, supplemented with 10%) fetal bovine serum, 1% glutamine, gentamicine (40 ⁇ g/ml), and hygromycin (400 ⁇ g/ml).
• H-RasV12 overexpressing senescent BJ cells were generated as in 20 .
• BrdU incorporation assays were carried at least a week after cultures had fully entered the senescent state, as determined by ceased proliferation, DDR activation, SAHF formation, and senescence- associated ⁇ -galactosidase expression.
• Ionizing radiation (IR) was induced by a high- voltage X-rays generator tube (Faxitron X-Ray Corporation). In general, cultured cells were exposed to 2 Grays for the foci formation assay. The authors used 5 Grays for the G2/M checkpoint assays and 10 Grays for the Gl/S checkpoint assays.
• Cherry-Lac and I-Sce I-restriction endonuclease expressing vector were transfected by lipofectamine 2000 (Invitrogen) in a ratio of 3: 1. 16h post transfection around 70% of the cells were scored positive for DDR markers at the Lac array.
• Dicer and Drosha knocked-down NIH2/4 cells were infected with Lentiviral particles carrying pLKO. l, shDicer or shDrosha vectors. After 48 hours cells were superinfected with Adeno Empty Vector or Adeno I-Sce I [Anglana et al. Nucl Ac Res 1999]. Nuclei were isolated the day after the adenoviral infection.
• ER-I-PpoI endonucleases Transient expression of ER-I-PpoI endonucleases in HeLa cells was carried out by Lipofectamine 2000 transfection and 16 hours later tamoxifen (0.1 ⁇ ) was added to culture medium to induce the activation of the endonuclease. 4 hours later cells were fixed for immunostaining or used for RNA extraction. Cherry-Lac transfected (mock) cells were used as control in these experiments.
• NIH2/4 cells where grown in DMEM, supplemented with 10% fetal bovine serum, 1% glutamine, gentamicine (40mg/ml), and hygromycin (400mg/ml).
• Cherry-Lac and I-Sce I-restriction endonuclease expressing vectors were transfected with Lipofectamine 2000 (Invitrogen) with a 3: 1 ratio.
• LNA were first boiled at 90°C for 5 minutes and quickly chilled at 4°C for 5 minutes and then added in different combinations to the Cherry-Lac and I-Sce I transfection mix, at the final concentration of 200pM. 24h post transfection cells were scored for DDR markers at the Lac array.
• T19 fibrosarcoma cells (van Steensel, Cell 1998) were grown in DMEM supplemented with 10% fetal bovine serum, 1% glutamine and doxycycline (100 ng/ml). For induction, cells were grown without doxycycline for at least 7-8 days.
• CRE-ER TRF2 flox/flox MEFs (Lazzerini Denchi and de Lange, Nature 2007) were grown in DMEM supplemented with 10% fetal bovine serum and 1% glutamine.
• cells were grown in presence of 4-hydroxytamoxifen (600 nM) for 48 hours.
• BrdU incorporation cells were labeled with 10 ⁇ g/ml bromodeoxyuridine (BrdU, Sigma) for 16 hours and incorporation was evaluated by immunofluorescence after DNA denaturation.
• Antibodies Mouse anti-yH2AX, anti-H3K9me3, rabbit polyclonal anti-PH3 (Upstate Biotechnology); anti-pS/TQ (Cell Signaling Technology); anti-H2AX, anti-H3 and anti DICER (13D6) (Abeam); rabbit polyclonal anti-53BPl (Novus Biological) and mouse monoclonal anti-53BPl (a gift from Thanos Halazonetis); anti-MREl l (a gift from S.
• Cells were grown on poly-D-lysinated coverslips (poly-D- lysine was used at 5( ⁇ g/ml final concentration) and plated (15-20xl0 3 cells/cover) one day before staining. DDR and BrdU staining was performed as in 20 . Cells were fixed in 4% paraformaldehyde or methanol: acetone 1 : 1. NIH2/4 mouse cells were fixed by 4% paraformaldehyde as in 35 . Images were acquired using a wide field Olympus Biosystems Microscope BX71 and the analySIS or the MetaMorph software (Soft Imaging System GmbH).
• Comparative immunofluorescence analyses were performed in parallel with identical acquisition parameters; at least 100 cells were screened for each antigen. Cells with more than 2 DDR foci were scored positive. Foci intensity quantifications were performed using Cell Profiler software 2.0. Confocal sections were obtained with a Leica TCS SP2 AOBS confocal laser microscope by sequential scanning.
• Immunofluorescence for the experiments in Figs. 35-36.
• Cells were fixed with 1 : 1 methanol/acetone solution for 2 minutes at room temperature, or 4% paraformaldehyde for 10 minutes at room temperature. After blocking, cells were stained with primary antibodies for lh at room temperature, washed and incubated with conjugated secondary antibodies for 40 minutes at RT. Nuclei were stained with DAPI (1 ⁇ g/ml).
• Flag-DICER, Flag-DICER44ab and Flag-DICERl lOab were a kind gift of R. Shiekhattar.
• pLKO.l shDICER expressing vector was a kind gift of WC. Hahn.
• Short hairpin sequence for DICER is: CCG GCC ACA CAT CTT CAA GAC TTA ACT CGA GTT AAG TCT TGA AGA TGT GTG GTT TTT G (SEQ ID NO: l).
• pRETROSUPER shp53 as in 20 Short hairpin sequence for p53 was: AGT AGA TTA CCA CTG GAG TCT T (SEQ ID NO:2).
• Cherry-Lac-repressor and I-Sce I-restriction endonuclease expressing vectors were kind gifts of E. Soutoglou 35 .
• ER-I-Ppo I-restriction endonuclease expressing vector was a kind gift of Michael Kastan 33 .
• shRNA against mouse Dicer and Drosha expressing vectors were a kind gift of W.C. Hahn.
• shRNA for mouse Dicer CCG GGC CTC ACT TGA CCT GAA GTA TCT CGA GAT ACT TCA GGTCAA GTG AGG CTT TTT (SEQ ID NO:3).
• siRNA for mouse Drosha: CCG GCC TGG AAT ATG TCC ACA CTT TCT CGA GAA AGT GTG GAC ATA TTC CAG GTT TTT G (SEQ ID NO:4).
• siRNA The DHARMACON siGENOME SMARTpool siRNA oligonucleotide sequences for human 53BP1 , ATM, DICER, DROSHA were:
• GGA CAA GUC UCU CAG CUA U SEQ ID NO:6
• GAU AUC AGC UUA GAC AAU U SEQ ID NO:7;
• CAA CAU AGA CUA CAC GAU U (SEQ ID NO: 17);
• the DHARMACON siGENOME si RNA sequences for Human TNRC6A, B and C were: GW182/TNRC6A:
• siRNA against human DICER 3' UTR siRNA against human DICER 3' UTR
• AAC ACU UGU CAC UAC UUU CUC (SEQ ID NO:28).
• siRNAs were transfected by Oligofectamine (Invitrogen) at a final concentration of 200 nM in OIS cells and ⁇ in HNF.
• Oligofectamine Invitrogen
• siRNA transfection with deconvolved siRNA oligos the authors used 50 nM for smart pools and 12.5 nM for deconvolved siRNAs.
• RNA samples were isolated from cells using TRIzol (Invitrogen) or RNAeasy kit (Qiagen) according to the manufacturer's instructions, and treated with DNAse before reverse transcription. For microRNA isolation the authors used mzVVanaTM miRNA Isolation Kit (Ambion). cDNA was generated using the Superscript II Reverse Transcriptase (Invitrogen). cDNA was used as template in TaqMan® Gene Expression Assays (Applied Biosystems) for the evaluation of DICER (Assay ID: Hs00998580_ml) and DROSHA (Assay ID: Hs01095030_ml) mRNA levels.
• DICER Assay ID: Hs00998580_ml
• DROSHA Assay ID: Hs01095030_ml
• TaqMan® MicroRNA Assays were used for the evaluation of mature miR-21 and rnu44 and rnul9 expression levels (Assay ID: 000397, 001094 and 001003). 18S or ⁇ -actin was used as a control gene for normalization. miR21 and rnu44 enrichment in the small RNA-enriched fraction was evaluated as the ratio between PCR cycles (ct) for miR-21 or rnu44 and for ⁇ -actin mRNA after normalization to the same ratio in total RNA fraction.
• Real-time quantitative PCR reactions were performed on an Applied Biosystems ABI Prism 7900HT Sequence Detection System or on a Roche LightCycler 480 Sequence Detection System. The reactions were prepared using SyBR Green reaction mix from Roche. Ribosomal protein P0 (RPPO) was used as a human and mouse control gene for normalization.
• RPPO Ribosomal protein P0
• Primer sequences for real-time quantitative PCR were:
• TGTGAGTCCAGGATCTGCTACTT (SEQ ID NO:40) (Reverse);
• NIH2/4 cells were incubated at this point also with 100 ⁇ mirin (SIGMA) or DMSO for 15 minutes. Then, RNase A-treated cells were incubated with total, small or gel extracted RNA, or the same amount of tRNA, for additional 15 minutes at room temperature. If using mirin, NIH2/4 cells were incubated with total RNA in the presence of 100 ⁇ mirin or DMSO for 25 minutes at room temperature.
• RNA oligonucleotides were generated by SIGMA with a monophosphate modification at the 5' end.
• Sequences map to different regions of the integrated locus: two pairs map to a unique sequence flanking the I-Sce I restriction site (Oligo 1+Oligo 2 and Oligos 3+ Oligo 4), one to the Lac-operator (Oligo 5 + Oligo 6) and one to the Tet-repressor repetitive sequences (Oligo 7 +01igo 8). Two paired RNA oligonucleotides with the sequences of GFP were used as negative control (Oligo GFP 1+ Oligo GFP 2). Sequences are reported below.
• Oligo 1 5'-AUA ACA AUU UGU GGA AUU CGG CGC-3'(SEQ ID NO:45),
• oligo 2 5'-CGA AUU CCA CAA AUU GUU AUC C-3'(SEQ ID NO:46),
• oligo 3 5'-AUU UGU GGA AUU CGG CGC CUC UAG AGU CGA GG-3'(SEQ ID NO:47),
• oligo 4 5'- CCU CGA CUC UAG AGG CG-3'(SEQ ID NO:48),
• oligo 5 5'-AGC GGA UAA CAA UUU GUG GCC ACA UGU GGA-3'(SEQ ID NO:49)
• oligo 6 5'- UGU GGC CAC AAA UUG UU-3'(SEQ ID NO:50)
• oligo 7 5'-ACU CCC UAU CAG UGA UAG AGA AAA GUG AAA GU-3'(SEQ ID NO:51),
• oligo 8 5'-CUU UCA CUU UUC UCU AUC ACU GAU AGG GAG UG-3'(SEQ lD NO:52)
• GFP 1 5'-GUU CAG CGU GUC CGG CGA GUU-3'(SEQ lD NO:53),
• GFP 2 5'-CUC GCC GGA CAC GCU GAA CUU-3'(SEQ lD NO:54)
• RNAs were resuspended in 60m M KG, 6mM HEPES-pH 7.5, 0.2mM MgC12, at the stock concentration of 12.5 ⁇ , denatured at 95 °C for 5 minutes and annealed fo l Ominutes at room temperature.
• DICER RNA products were generated as follows. A 550 bp DNA fragment carrying the central portion of the genomic locus studied (three Lac repeats, the I-Sce I site and two Tet repeats) was flanked by T7 promoters at both ends and was used as a template for in vitro transcription with the TurboScript T7 transcription kit (AMSBIO). The 500nt long RNA obtained was purified and incubated with human recombinant DICER enzyme (AMSBIO) to generate 22-23nt RNAs. RNA products were purified, quantified and checked on a polyacrylamide or an agarose gel. As a control, the same procedure was followed with a 700bp construct containing the RFP DNA sequence. Equal amounts of DICER products generated in this way were used in complementation experiment in NIH2/4 cells following RNase treatment.
• AMSBIO TurboScript T7 transcription kit
• RNaseA treatment for the experiments in Figs. 35.
• CRE-ER TRF2 flox/flox MEFs (Lazzerini Denchi and de Lange, Nature 2007) were induced to generate TRF2 knockout and telomere uncapping.
• 48 hours later cells were permeabilized with 0.6% Tween 20 in PBS for 15 min at room temperature.
• R ase A treatment was carried out in 1ml of 1 mg/ml ribonuclease A from bovine pancreas (Sigma-Aldrich catalogue no. R5503) in PBS for 30 minutes at room temperature.
• LNA Transfection for the experiments in Fig. 36. LNA were first boiled at 90°C for 5 minutes, chilled at 4°C for 5 minutes and transfected with Lipofectamine RNAiMAX (Invitrogen) at the final concentration of 200 nM.
• Lipofectamine RNAiMAX Invitrogen
• RNA preparation Small RNA preparation.
• Total RNA was isolated from cells using TRIzol (Invitrogen) according to the manufacturer's instructions.
• TRIzol Invitrogen
• the mzVVana microRNA isolation kit employs an organic extraction followed by immobilization of RNA on glass-fiber (silica- fibers) filters to purify either total RNA, or RNA enriched for small species.
• RNA extraction ethanol is added to samples, and they are passed through a Filter Cartridge containing a glass-fiber filter, which immobilizes the RNA.
• RNA is eluted with a low ionic-strength solution.
• ethanol is added to bring the samples to 25% ethanol.
• this lysate/ethanol mixture is passed through a glass-fiber filter, large RNAs are immobilized, and the small RNA species are collected in the filtrate.
• the ethanol concentration of the filtrate is then increased to 55%, and it is passed through a second glass-fiber filter where the small RNAs become immobilized.
• This RNA is washed a few times, and eluted in a low ionic strength solution.
• an RNA fraction highly enriched in RNA species ⁇ 200 nt can be obtained 25 ' 66 .
• RNA extraction from gel Total RNA samples were heat-denatured, loaded and resolved on a 15% denaturing acrylamide gel [IX TBE, 7 M urea, 15% acrylamide (29: 1 acrykbis- acryl)]. Gel was run for 1 hour at 180 V and stained in GelRed solution. Gel slices were excised according to the molecular weight marker, moved to a 2 ml clean tube, smashed and RNA was eluted in 2 ml of ammonium acetate 0.5 M, EDTA 0.1 M in RNase-free water, rocking overnight at 4°C.
• WI38, BJ and MRC-5 cells were irradiated with lOGy and 1 hour afterwards incubated with BrdU for 7h; HCT116 and R O cells were irradiated 2Gy and incubated with BrdU for 2h.
• Cells were fixed with 4% paraformaldehyde and probed for BrdU immunostaining. At least 100 cells per condition were analyzed.
• HEK 293 calcium phosphate transfected cells were irradiated with 5 Gy and allowed to respond to IR-induced DNA damage in a cell culture incubator for 12, 24 or 36 hours. Then, at these three time points post irradiation, together with not irradiated cells, 1X10 6 cells were collected for Fluorescence Activated Cell Sorting (FACS) analysis, fixed in 75% ethanol in PBS, 30 minutes on ice. Afterwards, cells were treated 12 hours with 40 ⁇ g/ml of RNase A and incubated at least lh with propidium iodide (PI). FACS profiles were obtained by the analysis of at least 5X10 5 cells. In the complementation experiments HEK 293 were transfected using Lipofectamine RNAi Max (Invitrogen) and 48 hours later irradiated with 5 Gy. Cells were then treated as explained above.
• FACS Fluorescence Activated Cell Sorting
• zebrafish immunoblotting protein analysis 72 hours post fertilization (hpf) larvae were deyolked in Krebs Ringer's solution containing ImM EDTA, 3mM PMSF and proteases inhibitor (Roche complete protease inhibitor cocktail). Embryos were then homogenized in SDS sample buffer containing ImM EDTA with a pestle, boiled 5 min and centrifuged 13000 rpm for 1 min.
• Protein concentration was measured with the BCA method (Pierce) and proteins (50 ⁇ g-900 ⁇ g) were loaded in an SDS-12% (for ⁇ 2 ⁇ and H3) and SDS-6% polyacrylamide gel (for pATM and ATM), transferred to a nitrocellulose membrane, and incubated with anti-yH2AX (1 :2000, a gift from J. Amatruda 67 ), H3 (1 : 10000, Abeam), pATM (1 : 1000, Rockland), ATM (1 : 1000, Sigma). Immunoreactive bands were detected with horseradish peroxidase-conjugated anti-rabbit or anti-mouse IgG and an ECL detection kit (Pierce, Springfield, IL, USA). Protein loading was normalized to equal amounts of total ATM and H3.
• oligonucleotides carrying the binding sites for miR126 used for construction of pCS2:RFPmiR126 sensor are:
• RNA encoding for RFPmiR126 sensor was injected alone or in combination with Dicerl morpholino at a concentration of 10 pg/nl. Dicer morpholino was injected at a concentration of 5 ng/nl, and a volume of 2 nl/embryo.
• the authors injected donor embryos with a mixture of dicerl morpholino and mRNA encoding for GFP (5 pg/nl). Approximately 20 cells were transplanted from donor embryos at dome (5 hpf) stage to uninjected host at the same stage.
• RNA sequencing Nuclear RNA shorter than 200 nt was purified using mzVVanaTM microRNA Isolation Kit. RNA quality was checked on a small RNA chip (Agilent) before library preparation (Supplementary Figure 23 a). For Illumina hi Seq Version3 sequencing, spike RNA was added to each RNA sample in the RNA : spike ratio of 10,000 : 1 before library preparation and libraries for Illumina GA IIX were prepared without spike. An improved short R A library preparation protocol was used to prepare libraries 68 .
• adenylated 3' adapters were ligated to 3' ends of 3 ' -OH short R As using a truncated RNA ligase enzyme followed by 5' adapter ligation to 5 '-monophosphate ends using RNA ligase enzyme, ensuring specific ligation of undegraded short RNAs.
• cDNA was prepared using a primer specific to the 3'adapter in the presence of Dimer eliminator and amplified for 12-15 PCR cycles using a special forward primer targeting the 5' adapter containing additional sequence for sequencing and a reverse primer targeting the 3' adapter.
• the amplified cDNA library was run on a 6% polyacrylamide gel and the 100 bp band containing cDNAs up to 33 nt was extracted using standard extraction protocols. Libraries were sequenced after quality check on a DNA high sensitivity chip (Agilent). Multiplexed barcode sequencing was performed on Illumina GA-IIX (35 bp Single end reads) and Illumina Hi seq version3 (51 bp single end reads). Sequences of all the DDRNAs identified in this study will be available for free downloading by the time of publication at short read archive.
• Results are shown as means plus/minus standard error (s.e.m.). p- value was calculated by Chi-squared test. QRT-PCR results are shown as means of a triplicate plus/minus standard deviation (s.d.) and p-value was calculated by Student's t- test as indicated. * indicates p-value ⁇ 0.05.
• the differences in the fraction of 22-23 nt vs total short RNAs at the locus between the wildtype, Dicer knockdown, and Drosha knockdown before and after cut was calculated by fitting a negative binomial model to the sRNA count data and performing a likelihood ratio test, keeping the fraction of 22-23 nt vs total short RNAs at the locus fixed across conditions under the null hypothesis and allowing it to vary between conditions under the alternative hypothesis.
• LNA 1 TTATCCGCTCACAATTCCACAT (SEQ ID NO:57)
• LNA 2 ATGTGGAATTGTGAGCGGATAA (SEQ ID NO:58)
• LNA 3 (Cntrl in Fig. 36): ACTGATAGGGAGTGGTAAACT (SEQ ID NO:59)
• LNA 4 AGAGAAAAGTGAAAGTCGAGT (SEQ ID NO:60)
• LNA 5 control in Fig. 34: CCCTAACCCTAACCCTAACCC (SEQ ID NO:61)
• LNA 6 GGGTTAGGGTTAGGGTTAGGG (SEQ ID NO:62)
• Oncogene-induced senescence is a non-proliferative state characterized by a sustained DDR 20 ' 21 (caused by high level of endogenous DNA damage) and senescence- associated heterochromatic foci (SAHF) 22 . Since the RNAi-machinery has been involved in heterochromatin formation 23 , the authors investigated whether the inactivation of components of the RNAi machinery could have an impact on escape from senescence induced in human fibroblasts by transduction of H-RasV12 (referred here as OIS cells).
• DDR plays a crucial role in the maintenance of the proliferative arrest in OIS cells 20 ' 21 .
• the authors monitored whether DICER or DROSHA inactivation had an impact on DDR foci maintenance.
• the authors therefore stained cells for markers of active DDR such as the autophosphorylated form of ATM (pATM), phosphorylated substrates of ATM and ATR (pS/TQ), 53BP1 and ⁇ 2 ⁇ .
• the authors observed that DICER or DROSHA inactivation significantly reduces the number of 53BP1, pATM and pS/TQ foci positive cells ( Figures la, b and 9a) even though 53BP1, ATM or H2AX protein levels are not reduced ( Figure 9c).
• DICER or DROSHA inactivation impairs ionizing radiation-induced DDR foci formation.
• DICER and DROSHA in DDR activation are specific for the senescence condition or whether DICER or DROSHA inactivation has also an impact on ionizing radiation (IR)-induced DDR activation in proliferating non-senescent cells. Therefore, the authors transiently inactivated DICER or DROSHA by a pool of siRNA in human normal fibroblasts (HNF - WI38; Figure 12a and b), exposed them to IR and a few hours later the authors stained them for markers of activated DDR.
• HNF - WI38 human normal fibroblasts
• DNA damage elicits DDR signal transduction leading to checkpoint-dependent cell-cycle arrest at two critical transition steps: the Gl/S checkpoint and the G2/M checkpoint 1 .
• DICER irradiated empty- vector (EV) transfected cells progressively accumulate in the G2 phase of the cell cycle, as a consequence of the checkpoint enforcement.
• DICER as well as p53 knocked-down cells, did not arrest upon DNA damage and passed through the G2/M transition ( Figure 2 c and d).
• DDR foci are sensitive to RNase A and DICER and DROSHA RNA products allow DDR foci reformation.
• RNA in DDR activation IR-exposed human cells (HeLa) were permeabilized by a mild detergent and treated with the broad-specificity RNA nuclease RNase A.
• IR may induce different kinds of DNA lesions
• RNA molecules involved in DDR focus reformation To gauge the length of the RNA molecules involved in DDR focus reformation, the authors enriched total HeLa RNA for short RNAs by chromatography ( ⁇ 200nt; Figure 25 a) and the authors used proportional volumes of total and short RNA to restore DDR foci in RNase A treated cells. The authors observed that the short RNAs-enriched fraction was sufficient to restore pATM, pS/TQ and 53BP1 foci indicating that this fraction contains the active RNA molecules ( Figure 25b, c). To attain better RNA size separation, the authors resolved total RNA on a polyacrylamide gel and recovered RNAs of different lengths: longer than lOOnt, between lOOnt and 35nt and between 35nt and 20nt ( Figure 4c and 26).
• RNA components required for DDR foci assembly are short and in the size range of the RNA products generated by DICER and DROSHA.
• DICER RNA products directly contribute to DDR foci formation. To do so, the authors extracted total RNA from wild-type and D/CEi? exo " 5 -hypomorphic cells and the authors used these two RNA preparations to restore DDR foci in RNase A- treated irradiated cells. Total RNA preparations from the two cell lines are expected to have the same composition apart from the population of DICER RNA products 25 .
• RNA extracted from wild-type cells does restore pATM, pS/TQ or 53BP1 foci
• RNA extracted from DICER exon5 hypomorphic cells does not ( Figure 4d, e).
• RNA from DICER exon5 hypomorphic cells transfected with a vector expressing wild-type but not mutant DICER allows DDR foci reformation ( Figure 27).
• RNA purified from DROSHA-inactivated cells is unable to restore DDR foci
• the authors knocked-down DROSHA, and GFP as control, by siRNA in HeLa cells, purified RNA and used these RNA preparations to attempt to restore DDR foci.
• DDR focus formation at a defined damaged genomic site requires damage site- specific RNAs.
• Ionizing radiations induce the formation of DNA lesions that are heterogeneous in nature and random in their location.
• the authors studied a single DSB at a unique, defined and traceable genomic locus. The authors therefore took advantage of NIH2/4 mouse cells carrying an integrated copy of the I-Sce I restriction site flanked by an array of Lac-repressor (Lac) binding sites and Tet repeats 35 .
• the expression of the I-Sce I restriction enzyme together with the fluorescent protein Cherry-Lac-repressor (Cherry-Lac) allows the visualization in the nucleus of the site-specific DSB generated by the nuclease.
• RNA was added to the RNase A-treated samples NIH2/4 cells re-acquired a bright 53BP1 focus co-localizing with the Cherry-Lac-repressor in a manner dependent on the RNA amount used ( Figure 5a and b). Therefore, the very same DDR focus generated on a defined DSB can disassemble and reassemble in a manner dependent on RNA.
• RNA extracted from either cell lines to attempt to restore 53BP1 focus formation in RNase A-treated NIH2/4 cells that had experienced the I- See I-induced DSB: the two RNA preparations are expected to differ only in the potential presence of RNA transcripts generated from the exogenous integrated construct carrying the Lac and Tet repeats and the I-Sce I site.
• MRE11/RAD50/NBS1 (MRN) complex is a key DNA damage sensor and a necessary cofactor of the apical DDR regulator ATM ⁇
• MRE11 focus formation upon I-Sce I induction is sensitive to RNase A-treatment ( Figure 29a).
• mirin a specific small molecule inhibitor of MRN 36 which, as expected, prevents ATM activation also following I-Sce I induction ( Figure 29b).
• the authors therefore tested whether RNAs involved in DDR modulation engage MRN.
• the authors observed that in the presence of mirin, NIH2/4 RNA is unable to induce 53BP1 or pATM focus reformation ( Figure 5d and e). This result demonstrates that RNAs at sites of DNA damage modulate DDR in a MRN- dependent manner.
• RNAs originating from the integrated locus To detect potential short RNAs originating from the integrated locus, the authors isolated nuclear RNA from parental NIH 3T3 cells transfected with the I-Sce I (mock), NIH 2/4 cells transfected with Cherry-Lac-repressor (uncut) and from NIH 2/4 cells transfected with the I-Sce I (cut) and further selected them for length ( ⁇ 200 nt) - this procedure enriches for RNAs active in DDR foci reformation 40 folds (data not shown). Libraries prepared from these samples were sequenced by Illumina GAII-X to obtain 15-32bp cDNA reads ( Figure 30a-d).
• RNAs are biologically active both when added to the cells together with total RNA from parental cells (cells not carrying the integrated endogenous locus; Figure 6a) or with yeast tRNA ( Figure 30f), thus in the absence of any additional mammalian RNA.
• RNAs generated at the locus were performed deeper sequencing experiments of nuclear RNAs ⁇ 200 nt from wildtype NIH 2/4 samples before and after cut using the Illumina Hi seq Version3 ( Figure 31). To study the biogenesis of these RNAs, the authors also sequenced ⁇ 200 nt nuclear RNAs from NIH 2/4 cells following Dicer or Drosha knockdown (WT uncut, Dicer KD uncut, Drosha KD uncut, WT cut, Dicer KD cut, Drosha KD cut) ( Figure 32a). To ease normalization, each RNA preparation was spiked with a short RNA "spike" before library preparation.
• the authors compared the fraction of 22-23 nt vs total RNAs at the locus to the same fraction at non-miRNA genomic loci - at such loci, any 22-23 nt RNAs are most likely products of random degradation or Dicer/Drosha independent enzymatic processing.
• RNAs are the bulk of the RNA species detected at the locus, they depend on Dicer and, to an extent, on Drosha, and their proportion increases upon DNA damage. Their unlikelihood to be random degradation products is further indicated by their differential abundance compared to the rest of non-miRNA loci and the observed 5' and 3' base bias.
• LNAs Sequence-specific inhibitory oligonucleotides
• DDRNAs identified by deep sequencing are biologically active and have a causative role in sequence-specific DDR focus reformation at the damaged site, following removal of all cellular RNAs by RNaseA treatment (Fig. 6a; Fig. 30f).
• LNA Locked Nucleic Acids
• Cells carrying the integrated locus were co-transfected with Cherry-Lac and I-Sce I- restriction endonuclease-expressing vectors and with either no LNA (sample 1), control LNA carrying an unrelated sequence which is not part of the locus (sample 2) or different sets of LNA (samples 3-7) (Fig. 34b). 24 hours post transfection, cells were fixed and stained for DDR markers. The authors analyzed the samples at the confocal microscope and scored as positive those cells that showed a DDR signal at the locus. As shown in Figure 34b, the portion of cells showing a specific ⁇ 2 ⁇ focus co-localizing with the Cherry-Lac signal was not significantly affected by the transfection of any LNA.
• RNA-specific inhibitory oligonucleotides i.e. LNAs
• LNAs Long RNA-specific inhibitory oligonucleotides
• DDRNAs short RNAs with the sequence of a damaged locus
• Figs. 4,5 endonuclease cleavage
• telomere uncapping causes telomere uncapping, so telomeres are recognized as DNA DSBs. This may lead to DDR activation, cellular senescence, chromosomal fusions and genome instability (Sfeir and de Lange, Science 2012).
• TRF2 flox/flox mouse embryonic fibroblasts (MEFs) (Lazzerini Denchi and de Lange, Nature 2007).
• MEFs CRE-ER TRF2 flox/flox mouse embryonic fibroblasts
• TRF2 ⁇ ⁇ TRF2-knockout
• DDRNAs with telomeric sequence are generated and they are necessary for DDR cascade activation.
• telomeres can accumulate damaged telomeres during ageing, due to telomeric shortening (Harley, Nature 1990; Herbig, Mol Cell 2004; d'Adda di Fagagna, Nature 2003) or to endogenous or exogenous DNA damage occurred at telomeres. DNA damage accumulate and DDR signaling persists at telomeres as they are not repairable (Fumagalli, Nat Cell Biol 2012). In both cases this persistent DDR activation at telomeres leads to cellular senescence. If DDRNAs are necessary for DDR at damaged telomeres, inhibiting their action could suppress DDR activation and potentially prevent or revert the senescence phenotype.
• T19 fibrosarcoma cells that express an inducible dominant negative (DN) allele of flag-tagged TRF2 (van Steensel, Cell 1998).
• DN inducible dominant negative
• telomeres are dysfunctional and cells show a strong DDR activation at telomeres (data not shown and van Steensel, Cell 1998).
• Induced cells, in which Flag-DN TRF2 was expressed, are positive for Flag immunostaining (Flag +).
• DDRNAs DDR-regulating RNAs
• Oncogene activation can trigger DDR and DDR-induced cellular senescence acts as tumor suppressive mechanism 2 ' 37 .
• DICER inactivation enhances tumor development in a K-Ras- induced mouse model of lung cancer 38 ' 39 and inactivation of various components of DICER and DROSHA complexes stimulate cell transformation and tumorigenesis 38 .
• mutations of DICER and TARBP2 a DICER co factor affecting its stability, have been described in human carcinomas 40 ' 41 .
• individual microRNAs have been reported both to promote and to reduce cell proliferation by regulating stability and translation of mRNAs encoding proteins with different roles in cell proliferation 18 : it is therefore presently unclear how RNAi apparatus inactivation favors tumorigenesis.
• DDRNAs are locally generated and favor the assembly of DDR factors in the shape of detectable DDR foci at the DNA damaged site.
• RNA sequencing confirmed the presence of short RNAs arising from the integrated exogenous locus which are induced upon cut. Comparison with short RNAs generated at other non miRNA genomic loci indicates that they are distinct from products of RNA degradation and their nucleotide bias at 5 ' end and 3 ' end indicates that these RNAs are processed at preferential RNA precursors sites.
• RNA molecules in DDR display genetic interactions with the RNAi machinery 56 and components of the large DROSHA complex have been identified in a ATM-dependent phosphoproteome screen 57 .
• RNAi pathway 58 In Drosophila, repeated DNA integrity is dependent on RNAi pathway 58 .
• DICER is specifically cleaved by caspases during apoptosis 63 .
• ATM has been shown to directly modulate the biogenesis of DICER and DROSHA RNA products by phosphorylating KSRP 64 .

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